Methods of using myelin basic protein peptide compositions

ABSTRACT

The present invention relates to a composition which comprises the following myelin basic protein peptides: MBP 30-44; MBP 83-99; MBP 131-145; and MBP 140-154. The composition may be used to treat a disease, in particular multiple sclerosis and/or optical neuritis and the invention also relates to such uses and methods.

This application is a divisional of U.S. patent application Ser. No.12/740,976, having a 35 USC 371(c) date of Jul. 21, 2010, which is theU.S. National Stage of International Application No. PCT/GB2008/003673,incorporated by reference, filed Oct. 30, 2008, which claims prioritybenefit of Great Britain Application No. 0721430.7, filed Oct. 31, 2007and Great Britain Application No. 0800962.3, filed Jan. 18, 2008.

The present invention relates to a composition which comprises myelinbasic protein peptides. The composition may be used for the treatment ofmultiple sclerosis.

INTRODUCTION

Multiple Sclerosis

Multiple Sclerosis (MS) is the most common disabling neurologicalcondition affecting young adults. Around 85,000 people in the UK haveMS.

In multiple sclerosis (MS), inflammation of nervous tissue causes lossof myelin, a fatty material that acts as a protective insulation fornerve fibres in the brain and the spinal cord. This loss of myelin, ordemyelination, leaves multiple areas of scar tissue, or sclerosis, alongnerve cells. Consequently, the sclerosis results in multiple and variedneurological signs and symptoms, usually with repeated relapse andremission.

Common symptoms of MS include reduced or loss of vision, stumbling anduneven gait, slurred speech, as well as urinary frequency andincontinence. In addition, MS can cause mood changes and depression,muscle spasms and severe paralysis.

It is now generally accepted that MS is an autoimmune disease mediatedby autoreactive T-cells.

Current treatments for MS generally suppress the immune system. Forexample, one treatment includes transplantation of bone marrow alongwith administration of cytostatics and immunosupressive drugs. Thistreatment is effective for some patients, but it is expensive andrelatively high-risk. Additionally, the administration of cytostatics isconsidered controversial in treating MS because its effects are unclearand potential side-effects are severe.

Treatment with interferon-beta (IFNβ) reduces the symptoms of MS in somepatients and is therefore widely used. However, the mechanism of actionof interferon-beta is unclear and IFNβ treatment is ineffective for manypatients. Furthermore, treatment with IFNβ is compounded by thedevelopment of anti-IFNβ antibodies in most patients Giovannoni, G.,Munschauer, F. E., 3rd, and Deisenhammer, F. (2002). Neutralisingantibodies to interferon beta during the treatment of multiplesclerosis. J Neurol Neurosurg Psychiatry 73, 465-469.

Currently, an effective treatment for MS does not exist. Treatment isfocused on merely reducing its symptoms, usually by general suppressionof the immune system. There is thus an acute need for a therapy whichspecifically targets local immune responses associated with onset andprogression of the disease.

Synthetic Peptides

Metzler and Wraith (Int. Immunol. 5:1159-1165 (1993)) were the firstresearchers to describe the use of synthetic peptides to inducesuppression of an autoimmune response in the mouse experimentalautoimmune encephalomyelitis (EAE) model, a commonly used in vivo modelof MS. In this study, peptides derived from MBP were administered by theintranasal route, and it was found that the level of disease suppressioncorrelated with the antigenic strength of the peptide used.

Later, in 1995, Liu and Wraith (Int. Immunol. 7:1255-1263) showed thatit was also possible to induce suppression of EAE in mice by theintraperitoneal administration of soluble MBP-derived peptides. In thisstudy, suppression of both Th1 and Th2 responses was achieved, and itwas shown that administration of peptides after the start of an immuneresponse could lead to suppression of the on-going immune reaction.

However, it was found that not all peptides capable of acting as T-cellepitopes are capable of inducing tolerance. The myelin basic protein(MBP) peptide 89-101 is an immunodominant antigen after immunisation andis also a very effective immunogen both in terms of priming for T cellreactivity and induction of EAE. However, this peptide has been shown tobe ineffective at inducing tolerance when administered in solution(Anderton and Wraith (1998) Eur. J. Immunol. 28:1251-1261).

The present inventors have previously shown that there is a link betweenthe capacity of a peptide to bind to an MHC class I or II molecule andbe presented to a T cell without further antigen processing and itscapacity to induce tolerance in vivo. Peptides which are antigenprocessing independent (i.e. do not require further antigen processingto bind MHC) can be predicted to be tolerogenic in vivo. These peptideshave been termed “apitopes”, for Antigen Processing IndependentepiTOPES.

WO 02/16410 describes the following apitopes from myelin basic protein(MBP): 30-44, 80-94, 83-99, 81-95, 82-96, 83-97, 84-98, 110-124,130-144, 131-145, 132-146 and 133-147.

WO 03/064464 identifies the following MBP peptides as being apitopes:134-148; 135-149; 136-150; 137-151; 138-152 and 140-154.

SUMMARY OF THE INVENTION

The present inventors have now found that a “cocktail” of four MBPpeptides, all of which are apitopes, is particularly effective intreating MS. It is thought that the four peptides may exert asynergistic effect when in combination.

In a first aspect, therefore, the present invention relates to acomposition which comprises the following myelin basic protein peptides:

-   -   MBP 30-44;    -   MBP 83-99;    -   MBP 131-145; and    -   MBP 140-154.

The composition may consist essentially of MBP 30-44, 83-99, 131-145 and140-154.

The composition may be used for treating or preventing a disease, inparticular multiple sclerosis.

The composition may be used for treating or preventing optic neuritis,in particular optic neuritis associated with multiple sclerosis.

In a second aspect, the present invention relates to a method oftreating or preventing multiple sclerosis and/or optic neuritis byadministering a composition according to the first aspect of theinvention to a subject.

In the method of the second aspect of the invention the composition maybe administered following a dose-escalation protocol.

It has been found that two of the peptides in the cocktail are HLA-DQ6binding (MBP 30-44 and 131-145) and two are HLA-DR2 binding (MBP 140-154and 83-99). The combined use of these apitopes provides more widespeadcover of the different Major Histocompatibility Complex (MHC) haplotypesseen in MS patients than therapy with a single peptide.

The method of the second aspect of the invention may involveadministration of the composition to a HLA-DQ6 or HLA-DR2 positivesubject.

In a third aspect, the present invention provides a kit which comprisesthe following myelin basic protein peptides:

-   -   MBP 30-44;    -   MBP 83-99;    -   MBP 131-145; and    -   MBP 140-154        for simultaneous, separate or sequential administration.

INTRODUCTION TO FIGURES

FIG. 1: Presentation of MBP 30-44 by HLA-DQ6 to T-cell clone MS 49:D3

FIG. 2: Presentation of MBP 130-144 by HLA-DQ6 to T-cell clone MS 17:A2

FIG. 3: Presentation of MBP 139-153 by HLA-DR2a and HLA-DR2b transfectedL-cells to T-cell clone N5:19

FIG. 4: Proliferative response of lymph node cells followingtolerisation with Apitope MS6

FIG. 5: Proliferation of splenocytes in response to Apitope MS7.Splenocytes obtained from mice treated by intranasal administration ofeither PBS or Apitope MS7 (83-99)

FIG. 6: Proliferation of splenocytes in response to Apitope MS7.Splenocytes obtained from mice treated by subcutaneous/intradermaladministration of either PBS or Apitope MS7 (83-99)

FIG. 7: Production of IFNγ and IL-2 by splenocytes obtained from micetreated by subcutaneous/intradermal administration of either PBS orApitope MS7 (83-99)

FIG. 8: Example of Snellens chart

FIG. 9: Patient PBMC responses to human myelin basic protein (MBP)

FIG. 10: Patient PBMC responses to ATX-MS-1467

FIG. 11: Comparison of the T cell proliferative response to MBP prior to(visit 1) and following (visit 8) treatment with ATX-MS-1467

DETAILED DESCRIPTION

The first aspect of the invention relates to a composition whichcomprises a plurality of peptides from myelin basic protein.

Myelin Basic Protein

Myelin basic protein (MBP) is an 18.5 kDa protein isolatable from humanbrain white matter. The mature protein has 170 amino acids and thesequence is widely available in the literature (see for example: Chou etal (1986) J. Neurochem. 46:47-53, FIG. 1; Kamholz et al (1986), PNAS83:4962-4966, FIG. 2; U.S. Pat. No. 5,817,629, SEQ ID NO: 1; Roth et al(1987), J. Neurosci. Res. 17:321-328, FIG. 4; Medeveczky et al (2006),FEBS Letters 580:545-552, FIG. 3B).

MBP Peptides

The term “peptide” is used in the normal sense to mean a series ofresidues, typically L-amino acids, connected one to the other typicallyby peptide bonds between the α-amino and carboxyl groups of adjacentamino acids The term includes modified peptides and synthetic peptideanalogues.

The peptides used in the composition and kit of the present inventionmay be made using chemical methods (Peptide Chemistry, A practicalTextbook. Mikos Bodansky, Springer-Verlag, Berlin.). For example,peptides can be synthesized by solid phase techniques (Roberge J Y et al(1995) Science 269: 202-204), cleaved from the resin, and purified bypreparative high performance liquid chromatography (e.g., Creighton(1983) Proteins Structures And Molecular Principles, WH Freeman and Co,New York N.Y.). Automated synthesis may be achieved, for example, usingthe ABI 43 1 A Peptide Synthesizer (Perkin Elmer) in accordance with theinstructions provided by the manufacturer.

The peptide may alternatively be made by recombinant means or bycleavage from a longer polypeptide. For example, the peptide may beobtained by cleavage from full length MBP. The composition of a peptidemay be confirmed by amino acid analysis or sequencing (e.g., the Edmandegradation procedure).

The peptides used in the compositions and kits of the present inventionare as follows:

MBP 30-44:H-Pro-Arg-His-Arg-Asp-Thr-Gly-Ile-Leu-Asp-Ser-Ile-Gly-Arg-Phe-NH₂MBP 83-99:H-Glu-Asn-Pro-Val-Val-His-Phe-Phe-Lys-Asn-Ile-Val-Thr-Pro-Arg-Thr-Pro-NH₂MBP 131-145:H-Ala-Ser-Asp-Tyr-Lys-Ser-Ala-His-Lys-Gly-Phe-Lys-Gly-Val-Asp-NH₂MBP 140-154:H-Gly-Phe-Lys-Gly-Val-Asp-Ala-Gln-Gly-Thr-Leu-Ser-Lys-Ile-Phe-NH₂

The terms “MBP 30-44”, “MBP 83-99”, “MBP 131-145” and “MBP 140-154”encompasses modified peptides. For example the peptides may be mutated,by amino acid insertion, deletion or substitution, so long as the MHCbinding-specificity of the unmodified peptide is retained, together withits capacity to be presented to a T cell. The peptide may, for example,have 5, 4, 3, 2, 1 or 0 mutations from the unmodified sequence.

Alternatively (or in addition) modifications may be made withoutchanging the amino acid sequence of the peptide. For example, D-aminoacids or other unnatural amino acids can be included, the normal amidebond can be replaced by ester or alkyl backbone bonds, N-or C-alkylsubstituents, side chain modifications, and constraints such asdisulphide bridges and side chain amide or ester linkages can beincluded. Such changes may result in greater in vivo stability of thepeptide, and a longer biological lifetime.

Modification of epitopes may be performed based on predictions for moreefficient T-cell induction derived using the program “Peptide BindingPredictions” devised by K. Parker (NIH) which may be found athttp://www-bimas.dcrt.nih.gov/cgi-bin/molbio/ken_parker_comboform (seealso Parker, K. C et al. 1994.J. Immunol. 152:163).

MBP peptides may be formulated into the composition as neutral or saltforms. Pharmaceutically acceptable salts include the acid addition salts(formed with free amino groups of the peptide) and which are formed withinorganic acids such as, for example, hydrochloric or phosphoric acids,or such organic acids such as acetic, oxalic, tartaric and maleic. Saltsformed with the free carboxyl groups may also be derived from inorganicbases such as, for example, sodium, potassium, ammonium, calcium, orferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine and procaine.

Composition

The composition of the present invention may be for prophylactic ortherapeutic use.

The composition may by prepared as an injectable, either as liquidsolution or suspension; solid form suitable for solution in, orsuspension in, liquid prior to injection may also be prepared. Thepreparation may also be emulsified, or the peptides encapsulated inliposomes. The active ingredients may be mixed with excipients which arepharmaceutically acceptable and compatible with the active ingredient.Suitable excipients are, for example, water, saline (for example,phosphate-buffered saline), dextrose, glycerol, ethanol, or the like andcombinations thereof

In addition, if desired, the composition may contain minor amounts ofauxiliary substances such as wetting or emulsifying agents and/or pHbuffering agents. Buffering salts include phosphate, citrate, acetate.Hydochloric acid and/or sodium hydryoxide may be used for pH adjustment.For stabilisation, disaccharides may be used such as sucrose ortrehalose.

In the composition, the relative ratio of the peptides (MBP 30-44:MBP83-99:MBP 131-145:MBP 140-154) may be approximately 1:1:1:1.Alternatively the relative ratios of each peptide may be altered, forexample, to focus the tolerogenic response on a particular sub-set ofautoreactive T-cells or if it is found that one peptide works betterthan the others in particular HLA types.

After formulation, the composition may be incorporated into a sterilecontainer which is then sealed and stored at a low temperature, forexample 4° C., or it may be freeze-dried.

Conveniently the composition is prepared as a lyophilized (freeze dried)powder. Lyophilisation permits long-term storage in a stabilised form.Lyophilisation procedures are well known in the art, see for examplehttp://www.devicelink.com/ivdt/archive/97/01/006.html. Bulking agentsare commonly used prior to freeze-drying, such as mannitol, dextran orglycine.

The composition may be administered in a convenient manner such as bythe oral, intravenous (where water soluble), intramuscular,subcutaneous, sublingual, intranasal, intradermal or suppository routesor implanting (e.g. using slow release molecules).

The conposition may advantageously be administered via intranasal,subcutaneous or intradermal routes.

The method and pharmaceutical composition of the invention may be usedto treat a human subject. Typically, a physician will determine theactual dosage which will be most suitable for an individual subject andit will vary with the age, weight and response of the particularpatient.

In a preferred embodiment a “dose escalation” protocol may be followed,where a plurality of doses is given to the patient in ascendingconcentrations. Such an approach has been used, for example, forphospholipase A2 peptides in immunotherapeutic applications against beevenom allergy (Müller et al (1998) J. Allergy Clin Immunol. 101:747-754and Akdis et al (1998) J. Clin. Invest. 102:98-106).

Kits

Conveniently, the four MBP peptides may be administered together, in theform of a mixed composition or cocktail. However, there may becircumstances in which it is preferable to provide the peptidesseparately in the form of a kit, for simultaneous, separate, sequentialor combined administration.

For example, the kit may comprise the four peptides in separatecontainers, or two containers, each comprising two peptides. Thecontents of the containers may or may not be combined prior toadministration.

The kit may also comprise mixing and/or administration means (forexample a vapouriser for intranasal administration; or a syringe andneedle for subcutaneous/intradermal dosing). The kit may also compriseinstructions for use.

The pharmaceutical composition or kit of the invention may be used totreat and/or prevent a disease.

In particular, the composition/kit may be used to treat and/or preventmultiple sclerosis and/or optical neuritis.

Multiple Sclerosis

Multiple Sclerosis (MS) is the most common neurological disorder amongyoung adults (20 to 40 years old) affecting around 385,000 people inEurope and 300,000 in the USA. It is a chronic degenerative disease ofthe central nervous system in which gradual destruction of myelin occursin patches throughout the brain and/ or spinal cord, interfering withneural connectivity and causing muscular weakness, loss of coordinationand speech and visual disturbances.

After 10 years, about half of the individuals who are initiallydiagnosed with Relapsing-Remitting MS (RRMS) find that the frequency ofrelapses decreases but disability increases. This is known as SecondaryProgressive MS (SPMS). Some estimates are that within 25 years, about90% of people with RRMS will progress to the secondary-progressive type.As with RRMS, Secondary Progressive MS can vary widely. For somepatients, the increase or progression of disability is very gradual, andfor others it can occur more quickly. In general, however, recovery fromattacks become less and less complete, and symptoms tend to increase anddisability grows. Clinical attacks become less pronounced and remissionstend to disappear, but more CNS tissue has now been destroyed and thecumulative damage is more apparent on MRI's.

The composition of the invention may be used to treat a patient withrecent-onset MS, RRMS or SPMS.

The composition of the present invention may reduce or ameliorate one ormore of the symptoms of MS, which include reduced or loss of vision,stumbling and uneven gait, slurred speech, urinary frequency andincontinence, mood changes and depression. SPMS can be associated withmuscle spasms and severe paralysis.

In particular the composition may ameliorate optical neuritis.

Optical Neuritis

Optic Neuritis (ON) is an inflammation, with accompanying demyelination,of the optic nerve serving the retina of the eye. It is a variablecondition and can present with any of the following symptoms: blurringof vision, loss of visual acuity, loss of some or all colour vision,complete or partial blindness and pain behind the eye.

Optic Neuritis is one of the most frequently presenting symptoms ofmultiple sclerosis and is the most common symptom at onset of MS. ONcan, however, be attributable to causes other than MS, such as ischemicoptic neuropathy.

ON presents unilaterally (in one eye only) in 70% of cases.

Most typically, Optic Neuritis first affects people aged between 15 and50 years of age. In this age group, studies indicate that more than 50%of patients will convert to Multiple Sclerosis within 15 years. As withMS, women are about twice as likely as men to present with ON and theprevalence in Caucasian peoples is higher than in other racial groups.

The main symptoms of Optic Neuritis are:

-   -   Loss of visual acuity (blurring of vision).    -   Eye pain.    -   Dyschromatopsia (reduced colour vision).    -   Movement and sound phosphenes (visual flashing sensations        brought about by side-to-side eye movement or sound).    -   Uhthoffs symptom, the worsening of symptoms with heat or        exhaustion.

Treatment of ON with a composition according to the present inventionmay prevent, reduce or ameliorate any of these symptoms. In order tomonitor progression of ON, visual accuity may conveniently be measuredusing a Snellens chart.

EXAMPLES

The following examples serve to illustrate the present invention, butshould not be construed as a limitation thereof. The inventionparticularly relates to the specific embodiments described in theseexamples.

For the purpose of the Examples, the following names may be used for theMBP peptides:

MBP peptide Name in Examples (interchangeable) 30-44 ATX-MS-01 ApitopeMS1 83-99 ATX-MS-07 Apitope MS7 131-145 ATX-MS-04 Apitope MS4 140-154ATX-MS-06 Apitope MS6

The composition of four peptides is termed ATX-MS-1467

Example 1 Identification of HLA-DQ6 Restricted Myelin Basic ProteinT-Cell Epitopes

MHC Class II genes confer susceptibility to multiple sclerosis (Nepomand Ehrlich (1991) Ann. Rev. Immunol. 9: 493-525). Within Caucasians inNorthern and Central Europe, Australia and Northern America theassociation is linked to the MHC molecule HLA-DR2 (DR2a [DRB5*0101] andDR2b [DRB1*1501], Haines et al (1998) Human Mol. Genet. 7: 1229-1234).Although the DR2 and DQ6 alleles of MHC are found at different loci,there is significant linkage disequilibrium between the two alleles. Theconcordance between the two alleles is 99%, much greater than expectedif the alleles were randomly re-associated. Therefore the possibilityarises that certain T-cell epitopes associated with MS may be HLA-DQ6restricted rather than HLA-DR2 restricted.

The aim of this study was to identify whether human MBP T-cell epitopesare presented by the HLA-DQ6 molecule to T-cell clones isolated fromHLA-DR2 positive MS patients. T-cell activation was measured by T-cellproliferation using [³H]-Thymidine incorporation.

Peptide Antigens

MBP peptides 30-44, 130-144 and 139-153 were synthesized using L-aminoacids and standard F-moc chemistry on an Abimed AMS 422 multiple peptidesynthesiser.

Antigen Presenting Cells

L-cells transfected with either HLA-DQ6, HLA-DR2a or HLA-DR2b, or Mgarcells (EACC, Porton Down, UK) which express HLA-DQ, DP and DR were usedas APCs.

T-Cell Clones

T-cell clones MS 49:D3 and MS 17:A2 were generated from MS patients andclone N5:19 was generated from a normal individual. All three cloneswere isolated from DR2 positive individuals.

Antigen Presentation Assay

Antigen presenting cells were incubated with the various concentrationsof peptide and the appropriate T-cells. Proliferation, and thereforeactivation, of the T-cells was measured by [³H]-Thymidine incorporation,and expressed as the stimulation index (SI=corrected counts per minute(ccpm) culture containing peptide/ccpm culture without peptide).

Results

When peptide MBP 30-44 was presented by L cells transfected withHLA-DQ6, T-cell clone MS 49:D3 gave a very strong proliferative response(1.5 fold greater than to Mgar control cells) at the highest peptideconcentration (FIG. 1). When MBP 130-144 was presented by L cellstransfected with HLA-DQ6 an even stronger response was induced in MS17:A2 T-cells (3.24 fold increase in proliferation compared to Mgarcontrol cells—FIG. 2).

A third T-cell clone isolated from a DR2 individual N5:19 responded tothe major epitope MBP 140-154 and to a series of overlapping 15 merpeptides spanning this region (138-156). Peptide 139-153 stimulatedproliferation of the N5:19 T-cell clone when presented by HLA-DR2atransfected L-cells, but not HLA-DR2b transfected L-cells (FIG. 3).

Conclusion

T-cell clones isolated from HLA-DR2 positive individuals respond toHLA-DQ6 restricted MBP T-cell epitopes. This implies that theassociation of HLA-DR2 with multiple sclerosis is not confined to DR2restriction of MBP T-cell epitopes but may also be DQ6 restricted.

In the MBP composition of the present invention, two of the peptides areHLA-DQ6 binding (MBP 30-44 and 131-145) and two are HLA-DR2 binding (MBP140-154 and 83-99). Therefore, peptide therapy with these apitopes maybe directed to either HLA-DR2 or HLA-DQ6 individuals.

Example 2 Induction of Tolerance with Apitope MS6 in a HLA:DR2Transgenic Mouse

This study was designed to demonstrate that an apitope (Apitope MS6),when presented by an MHC Class II molecule, can induce immunologicaltolerance in a humanized mouse model of multiple sclerosis. Apitope MS6is an apitope selected from within T-cell epitopes of MBP correspondingto MBP 140-154, and is presented by MHC Class II on antigen presentingcells, and can stimulate T-cells without being processed (see WO03/064464). The mouse model used was transgenic for the human MHCmolecule HLA:DR2 (DRB1*1501) (Madsen et al (1999) Nature Genetics23:343-347).

The induction of anergy or changes in the CD4+ T-cell population in amouse after administration of an apitope may be monitored by a reductionin T-cell proliferation when challenged with the antigen in vivo.

Peptide Synthesis

Apitope MS6 (MBP peptide 140-154) was synthesised using L-amino acidsand standard F-moc chemistry on an Abimed AMS 422 multiple peptidesynthesiser.

Mice and Tolerance Induction

HLA:DR2 transgenic mice aged 8-12 weeks were used in the study. The micewere tolerised by pre-treatment with 100 μg of Apitope MS6 in 25 μl ofphosphate buffered saline (PBS) or 25 μl PBS alone, by intranasaladministration on days -8, -6 and -4 prior to immunisation on day 0.

Following tolerisation, mice were immunised subcutaneously with 100 μlof an emulsion containing an equal volume of Complete Freund's Adjuvant(CFA) and PBS containing 200 μg of Apitope MS6 and 400 μg heat-killedMycobacterium tuberculosis. A control group of mice, previouslytolerised with PBS intranasally, were immunised without peptide.

At 10 days post subcutaneous immunisation, draining popliteal andinguinal lymph nodes were removed and T-cell activation was assessed byassaying the proliferation of T-cells in response to variousconcentrations of Apitope MS6. Proliferation was measured by[³H]-Thymidine incorporation, and expressed as the stimulation index(SI=corrected counts per minute (ccpm) culture containing peptide/ccpmculture without peptide).

Results

Group A mice that were tolerised with PBS and then immunised withApitope MS6 responded to antigenic stimulation when re-challenged withApitope MS6 in a dose dependent manner (FIG. 4). With increasingconcentration of peptide the SI increased from a median of 2.5 to 10.All the mice in this group demonstrated that PBS administeredintranasally could not induce tolerance to Apitope MS6.

In contrast, intranasal pre-treatment with Apitope MS6 had a profoundeffect on the proliferative response of lymphocytes stimulated with thispeptide. Lymphocytes from Group B mice were unable to respond to anysignificant degree, even at the high peptide concentration of 150 μg/ml(SI median 3, FIG. 4). These data suggest that Apitope MS6 has inducedtolerance in lymphocytes from HLA-DR2 mice.

Lymphocytes extracted from mice which had been pre-treated and immunisedwith PBS (Group C) failed to show any response to Apitope MS6 (FIG. 4).This lack of response to the peptide within Group C confirms that theproliferative response seen in Group A was indeed a response toimmunisation with Apitope MS6.

Conclusion

These data support the hypothesis that an MBP peptide (Apitope MS6) thatdoes not require processing and binds to HLA:DR2 MHC Class II molecules,can induce tolerance when administered intranasally.

Example 3 Induction of Tolerance with Apitope MS7 (MBP 83-99) in HLA:DR2and T-Cell Receptor (MBP) 82-100 Double-Transgenic Mice

This study investigates the ability of Apitope MS7 (MBP peptide 83-99)to induce tolerance in double transgenic mice expressing HLA:DR2together with the T-cell receptor for the HLA: DR2 bound myelin basicprotein (MBP) 82-100 peptide.

Splenocytes from these double transgenic animals proliferate in vitro inresponse to Apitope MS7. Reduction or abrogation of the in vitrosplenocyte response to an apitope following its repeated administrationin vivo indicates that a state of tolerance has been achieved.

Tolerance induction has been attempted using both the intranasal andsubcutaneous/intradermal routes of apitope administration.

Tolerance Induction

Groups of 6 or 7 age (8-12 weeks) and sex matched double transgenic micewere used. In the first experiment one group was treated ten timesintranasally with 100 μg Apitope MS7 in 25 μl PBS at regular intervalsover a period of three weeks. The control group received PBS alone. Inthe second experiment the same amount of peptide was given bysubcutaneous/intradermal administration in 100 μl of PBS. The controlgroup received the same volume of PBS.

Proliferation Assay

Three days after the last administration of peptide or PBS, the spleenswere harvested and cell suspensions prepared. The splenocytes wereincubation with 0.5 or 5 μg/ml Apitope MS7, and assessed forproliferation by [³H]-Thymidine incorporation after 48, 72 and 96 hoursof culture. Results are expressed as stimulation indices (SI=counts perminute (cpm) of culture containing antigen/counts per minute withoutantigen).

Cytokine Measurement

In the second experiment, where tolerance induction was induced via thesubcutaneous/intradermal route, cell culture supernatants from thesplenocytes were collected at 48 and 72 hrs of culture, and assayed forthe presence of IFN-γ and interleukin-2 (IL-2) using an enzyme linkedimmunosorbant assay (ELISA).

Results

At all three time points investigated, splenocytes from mice treatedwith PBS via both the intranasal and subcutaneous/intradermal routeshowed strong proliferation to Apitope MS7, the magnitude of theresponse increasing with peptide concentration. A striking reduction inproliferation was observed in splenocyte cultures from mice treated withApitope MS7 by both routes of administration. As shown in FIG. 5, after72hrs the mean SI observed with cultures containing 5 μg/ml Apitope MS7was 17 in mice treated intranasally with Apitope MS7. In thecorresponding cultures from control mice treated with PBS a mean SI of89 was observed. Similarly, as shown in FIG. 6 administration of ApitopeMS7 by the subcutaneous/intradermal route resulted in a decrease in meanSI from 124 in the PBS treated animals to 49 in animals treated withApitope MS7.

The levels of IFNγ and IL-2 detected in culture supernatants from 48 hrcultures are shown in FIG. 7. The repeated treatment with peptide invivo is seen to bring about an impressive reduction in secretion of bothcytokines, paralleling the reduction in the proliferative response.

Conclusion

These results indicate that tolerance to Apitope MS7 can be successfullyinduced in a mouse model designed to mimic the human system and supportsits potential for use in multiple sclerosis therapy. Furthermore itsefficacy is retained when administered by the subcutaneous/intradermalroute, the route of choice for humans.

Although the in vivo efficacy of Apitopes MS6 and MS7 has beendemonstrated it is not at present possible to demonstrate the efficacyof Apitopes MS1 and MS4. This is because of the lack of transgenic miceexpressing the HLA-DQ6 molecule. Mice expressing the molecule have beencreated, but it has been found that they fail to generate CD4 T-cells inthe thymus and do not therefore mount an immune response to antigen inthe context of HLA-DQ6.

Example 4 Dose Escalation Study with a Composition of Four MBP Peptides

The combination of four peptides (MBP 30-44, 131-145, 140-154 and 83-99)was used in an open-label, dose-escalation study of ATX-MS-1467 inpatients with secondary progressive multiple sclerosis.

Patients were monitored at four weeks and three months after the finalstudy dose for visual acuity (i.e. reduction in optic neuritis),immunological parameters and inflammation in the CNS.

Example 4A Monitoring Visual Acuity

Optic Neuritis (ON) is an inflammation, with accompanying demyelination,of the Optic Nerve serving the retina of the eye. ON is one of the mostfrequently presenting symptoms of multiple sclerosis, and is the mostcommon symptom at onset of MS.

Typical symptoms of ON include: blurring of vision, loss of visualacuity, loss of some or all colour vision, complete or partial blindnessand pain behind the eye.

The effect of treatment with ATX-MS-1467 on optic neuritis resultingfrom the neuroinflammatory process involved in MS was investigated.ATX-MS-1467 was given following the dose-escalation protocol outlinedabove, then one month following treatment the patient's visual acuitywas tested using a standard Snellens chart (FIG. 8).

The results show a clinically significant improvement in visual acuityone month post treatment. This was demonstrated in analysis of theinitial visual acuity examination at screening compared to the one monthfollow up test. Initial screening sight measurements of 6/24 and 6/9 (inright and left eyes respectively) improved to 6/9 (right eye) and 6/6(left eye) post treatment. The patient's eyesight had previously beenunchanged for the past two years.

Example 4B Monitoring Immunological Parameters

As explained above, the effect of peptide therapy with a cocktailcontaining four apitope™ peptides from myelin basic protein(ATX-MS-1467) has been investigated on MS patients. Each patient wasscreened at entry into the trial, up to 14 days prior to the first dose(visit 1). The first dose of 25 μg of ATX-MS-1467 was given on visit 2,50 μg on visit 3, 100 μg on visit 4, 400 μg on visit 5 and 800 μg onboth visits 6 and 7. Further examinations were conducted at one monthand three month follow-ups (visits 8 & 9). The following tablesummarises the protocol and shows the number of visits to the clinicalmade by each patient along with the dose of peptide and blood sampling.

TABLE 1 VISIT DOSE OF ATX-MS-1467 NUMBER (μg) COMMENT 1 — Pre-screeningvisit. 50 ml blood sample collected for immunology 2 25 No immunologysample collected 3 50 50 ml blood sample for immunology 4 100 50 mlblood sample for immunology 5 400 50 ml blood sample for immunology 6800 50 ml blood sample for immunology 7 800 50 ml blood sample forimmunology 8 — 50 ml blood sample for immunology taken four weeks postvisit 7

Blood samples were collected at visits 1 and visits 3 to 9 and testedfor immune responses to various antigens including purified proteinderivative (PPD) of Mycobacterium tuberculosis, as a positive control,purified human myelin basic protein (MBP) and ATX-MS-1467. The immuneresponses to be measured include in vitro T-cell proliferation,secretion of cytokines into tissue culture supernatants and generationof cytokine RNA.

Results

A significant response to MBP was seen, with a peak in proliferation atvisit 3 (FIG. 9). This correlated with a peak in secretion of interferongamma. Importantly, however, the level of interferon gamma secretionfell after the third visit and was at background level by visit 8. IL-10levels did not change significantly until visit 7 at which point therewas a significant increase in secretion of this cytokine. Levels ofIL-4, IL-5 and TNF alpha were close to background in response to MBP atall time points.

No proliferative response or increased cytokine response was observed toATX-MS-1467 (FIG. 10).

Conclusion

A response to MBP was observed at visit three demonstrating a heightenedsecretion of IL-2 and interferon gamma, correlating with a peak inproliferation at this time point. This increase in response is thoughtto be attributable to the administration of peptides.

Importantly, however, the transient increase in cytokine secretion wasfollowed by a return to baseline levels of interferon gamma. There wasalso a significant increase in IL-10 secretion following the secondadministration of ATX-MS-1467 at the highest dose. It has previouslybeen reported that, in animal models, specific immunotherapy withsynthetic peptides is effective and results in the induction of IL-10secreting regulatory T cells. The induction of IL-10 secretingregulatory T cells in mice involves a transient response to peptideswith secretion of interferon gamma at low levels (Burkhart et al., 1999Int Immunol 11: 1625-1634; Sundstedt et al., 2003 J Immunol170:1240-1248). This is followed by a decrease in interferon gamma and aconcomitant increase in IL-10. The kinetics of cytokine secretiondisplayed following treatment with ATX-MS-1467 effectively reproducesthe pattern previously observed in experimental mice, suggesting thatATX-MS-1467 may induce IL-10 secreting regulatory T cells.

Example 4C Response to MBP is Significantly Downregulated by Treatmentwith ATX-MS-1467

Six patients with secondary progressive multiple sclerosis (SPMS) wereenrolled into, and completed, a Phase I/IIa clinical trial following thedose-escalation protocol given in Example 4B.

HLA-genotyping showed that the six patients enrolled in the trialrepresented a wide range of HLA haplotypes, including the MS associatedHLA-DR15 haplotype. The broad HLA-DRB1 distribution represented bypatients enrolled in this trial implies that ATX-MS-1467 will be safeand well tolerated in MS patients irrespective of their HLA-DR genotype.

Treatment of patients with ATX-MS-1467 did not generate anti-peptideantibodies. This indicates that the use of ATX-MS-1467 is safe andcorrelates with the lack of injection site complications or allergicmanifestations associated with the trial.

A thorough analysis of T cell responses showed that:

-   -   a) Treatment with ATX-MS-1467 did not have a non-specific,        immunosuppressive effect. This is clearly shown by the fact that        the immune response to purified protein derivative, an antigen        from mycobacterium tuberculosis, was maintained (data not        shown).    -   b) Treatment with ATX-MS-1467, using this protocol, did not lead        to an aggressive immune response either to ATX-MS-1467 or myelin        basic protein (data not shown).    -   c) Comparison of the T cell proliferative response prior to        (Visit 1) and following (Visit 8) treatment with ATX-MS-1467        revealed a significant reduction in the response to myelin basic        protein (FIG. 11). The three individuals who responded to MBP at        V1 all showed a reduction in the response to the protein by V8.        Taking all of the patients together there is evidence for a        significant reduction in the response to MBP from V1 to V8        (P=0.0313).        Materials And Methods        Formulations and Dosages

Each of the four peptides is manufactured independently under contractusing solid phase peptide synthesis, and purified using HPLC. They arestored lyophilised.

ATX-MS-1467 is a 1:1:1:1 mixture of Apitopes MS1, MS4, MS6 and MS7 inphosphate buffered saline for intradermal administration.

Two strengths of ATX-MS-1467, designated ATX-MS-1467A and ATX-MS-1467Bcontaining 4 mg/ml and 0.5 mg/ml of peptide, respectively were preparedto allow dose escalation. The regime employs five dose-escalatinginjections (25, 50, 100, 400 and 800 μg total dose) given 7 to 14 daysapart. The patients then receive a second 800 μg dose 7 to 14 daysfollowing the first 800 μg dose. After receiving all six doses of studymedication, the patient is assessed at four-weeks and at 3 months afterthe final study dose.

Visual Accuity Test

A standard size Snellens chart for testing at 6 metres is used, withback light illumination and patient sitting at 6 metres distance (FIG.8).

Each eye is examined separately for the lowest line on the chart thatthe patient is able to read. This is then denoted as Visualacuity=6/(the line which the patient has read).

Immunoassays

i) T-Cell Proliferation:

Cryopreserved PBMC are set up in 1 ml cultures containing 1.5×10⁶ cellsin α-MEM in 48 well tissue culture plates (Nunc International, Costar,Corning Inc. New York USA). Responses to MBP and peptide antigens atvarious concentrations are monitored over a period of 10 days. Controlwells contain no antigen. After 20 hrs or 2, 4, 6, 8 and 10 days ofculture, duplicate 100 μl aliquots of cell suspension are removed fromeach 1 ml culture to measure proliferation in response to antigen byuptake of [³H] thymidine.

ii) Measurement of Secreted Cytokines (IL-2 and IL-4, IL-5, IL-10, TNF-αand IFN): Cytometric Bead Array Assay:

Culture supernatant cytokine levels are determined using the CytometricBead Assay (Becton Dickenson Biosciences, Cowley, Oxford, UK) followingthe manufacturer's instructions. Following acquisition of sample datausing the FACS Calibur (BD Biosciences), results are generated ingraphical and tabular form using BD CBA software. The minimumquantifiable levels of cytokine are as follows: IL-2 and IL-4 2.6pgml-1, IL-5 2.4 pgml-1, IL-10 and TNF-α 2.8 pgml-1, IFN-γ 7.1 pgml-1.

Quantitative Real Time PCR to Measure RNA of Cytokines (IL-2, IL-10,IFN-γ and TNF-α)

For analysis of IL-2, IL-10, IFN-γ and TNF-α, PCR reactions are carriedout in a final volume of 20 μl containing cDNA, PCR buffer, 6.25 mMMgCl₂, 0.4 mM dNTP mix, forward and reverse primers (forward primerconcentrations: IL-2 300 nM, IL-10 600 nM, IFN-γ 600 nM, TNF-α 600 nM,reverse primer concentrations: IL-2 600 nM, IL-10 900 nM IFN-γ 900 nM,TNF-α 600 nM), 200 nM FAM-TAMRA probe and 0.05 Uμl-1 Platinum Taqpolymerase (Invitrogen). The cycling conditions are as follows: aninitial denaturation step at 94° C. for 30 s, followed by 35 cycles with15 s at 94° C. and 1 min at 60° C. For β-2 microglobulin, the PCR mixdescribed above is supplemented with 0.1 uM forward and reverse primers,3 mM MgCl2 and quantitated with SYBR Green I (Molecular Probes Inc.,X30000 dilution of stock). Following a 1 min denaturation step at 95°C., amplification continues with 35 cycles of 15 s at 94° C., 1 min at61 oC and 1 min at 72° C. The PCR reactions and fluorescence detectionof amplicons generated is performed on an Opticon 2 system (MJ Research,USA). Baseline fluorescence is established by taking measurements fromcycle 1 to 10. Ct values are calculated by determining the point atwhich the fluorescence exceeds 8-10 times the standard deviation of thebaseline. Samples are assayed in duplicate and copy number calculatedfrom a standard curve for each target DNA. Differences in RNA input copynumber and cDNA synthesis are corrected by normalizing cytokineexpression to the expression of β-2 microglobulin.

HLA Typing

Analysis of HLA gene expression is performed by a standard single-strandconformation polymorphism, polymerase chain reaction technique on DNAextracted from peripheral blood leukocytes. The HLA-type of each patientis used for interpretation of the results of immunological assays.

Serum Anti-Peptide Antibody Assays

96 well plates are coated with 1-10 μg/ml of each apitope MS1, MS4, MS6or MS7 at pH 9.6 overnight at 4° C. Plates are washed four times withphosphate-buffered saline, pH 7.2, 0.05% Tween (PBS-Tween) and the wellsblocked with 5% FCS in PBS for 1 h at room temperature. Sera are diluted1:100 in PBS-Tween and incubated in duplicate wells for one hour at roomtemperature. After 4 washes, Goat anti-human IgG-horseradish peroxidaseconjugate (Sigma) diluted 1:12 000 in PBS is added to each well and theplates incubated for one hour at room temperature. After 4 washes 0.4mg/ml of o-phenylenediamine dihydrochloride (Sigma) plus 30% hydrogenperoxide is added and incubated at room temperature for 15-20 min.Colour development is stopped with 2.0 M H₂SO₄ (50 μl) and opticaldensity values (ODs) measured at 490 nm with an ELISA plate reader.Results are reported as OD for 1:100 diluted sera.

MRI Scan

Inflammation in the CNS is investigated using a gadolinium-enhanced MRIscan. The volume and number of enhancing lesions is examined andcompared with the baseline scan.

Various modifications and variations of the described methods and systemof the invention will be apparent to those skilled in the art withoutdeparting from the scope and spirit of the invention. Although theinvention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in chemistry or biology orrelated fields are intended to be covered by the present invention. Allpublications mentioned in the above specification are hereinincorporated by reference.

The invention claimed is:
 1. A method of inducing IL-10-secretingregulatory T cells in a subject in need of same which comprises the stepof administering to the subject a composition of myelin basic proteinpeptides, wherein the myelin basic protein peptides of the compositionconsist of myelin basic protein peptides MBP 30-44, MBP 83-99, MBP131-145 and MBP 140-154.
 2. The method according to claim 1, wherein thecomposition further comprises a pharmaceutically acceptable excipient.3. The method according to claim 2, wherein the subject has multiplesclerosis.
 4. The method according to claim 2, wherein the subject hasoptic neuritis.
 5. A method according to claim 2, in which thecomposition is administered to the subject following a dose-escalationprotocol.
 6. A method according to claim 2, wherein the composition isadministered to an HLA-DQ6 or HLA-DR2 positive subject.
 7. A method ofinducing tolerance to myelin basic protein in a subject in need of samewhich comprises the step of administering to the subject a compositionof myelin basic protein peptides, wherein the myelin basic proteinpeptides of the composition consist of myelin basic protein peptides MBP30-44, MBP 83-99, MBP 131-145 and MBP 140-154.
 8. The method accordingto claim 7, wherein the composition further comprises a pharmaceuticallyacceptable excipient.
 9. The method according to claim 7, wherein thesubject has multiple sclerosis.
 10. The method according to claim 7,wherein the subject has optic neuritis.
 11. A method according to claim7, in which the composition is administered to the subject following adose-escalation protocol.
 12. A method according to claim 7, wherein thecomposition is administered to an HLA-DQ6 or HLA-DR2 positive subject.